ABSTRACT There are several safe candidate adjuvants that potentiate beneficial immunity following vaccination. However, single adjuvant formulations have performed quite modestly in terms of stimulating the desired blend of humoral versus cellular immunity and/or Th1 versus Th2 immunity. Combination adjuvants offer a possible way forward, but the mechanisms of action of such combinations remain poorly defined. Therefore, we will combine the FDA-approved adjuvant Alum (alhydrogel) with the CD1d-binding glycolipid adjuvant ?-galactosylceramide (?-GC) and delineate the mechanisms of action with a particular emphasis on CD1d-restricted Natural Killer T (NKT) cells and humoral immunity. Alum is safe and stimulates excellent Th2 but poor Th1 immunity. We discovered that the Th2 response to Alum depends in large part upon diverse TCR-expressing Type II NKT cells (dNKT) which recognize CD1d/glycolipid complexes but do not recognize the ?-GC adjuvant. The ?-GC adjuvant stimulates semi-invariant TCR-expressing Type I NKT cells (iNKT) and results in a mixed Th1/Th2 response. It is already known from Phase I clinical trials in patients with cancer that ?-GC is well-tolerated and safe. Furthermore, modification of the ?-GC structure can readily be employed to skew the Type I NKT- dependent Th1/Th2 balance. Our objective for this proposal is to test the central hypothesis that the combination of Alum and ?-GC leads to CD1d/glycolipid presentation and a coordinated dNKT and iNKT- driven mixed Th1/Th2 response against vaccine antigens. In Specific Aim 1, we will perform a series of in vitro studies in primary murine and human cells to determine how the adjuvant combination affects CD1d Ag presentation and activation and functional differentiation of NKT cells into different effector subsets. We will also examine the effect of adjuvant combination on class I and class II presentation of co-administered protein antigens. In Specific Aim 2, we will undertake a series of in vivo experiments in mice to examine the functional consequences of adjuvant combination for Ab-mediated protection against bacterial toxins. Experiments to analyze cellular as well as humoral immunity will be included as the project develops and in vivo toxicity (or lack thereof) will be determined by measurement of pro-inflammatory cytokines as well as markers of autoimmunity and organ damage. We feel this project is innovative because it will provide the vaccine community with mechanistic information when CD1d-binding adjuvants are combined with Alum adjuvant and give careful consideration to NKT cell- driven components of the immune response. We feel the project is significant because it will illuminate potential avenues for inclusion of CD1d-binding glycolipids in mixed adjuvant platforms.